Project Summary Alternative polyadenylation (APA) generates highly diverse transcriptome and proteome in the brain to maintain proper neurological functions. Recent works from others and us revealed a complex and dynamic APA landscape in the brain with many cell type-specific APA regulations and an emerging role of APA in neurological disease. However, APA alterations in neurological disorders remain largely unexplored and rigorously designed in vivo high-throughput APA studies in brain disease are especially lacking due to technical difficulties. We broke the technical barrier in 2017 by developing the cTag-PAPERCLIP technology, which simultaneously profiles more than 20,000 poly(A) sites in individual cell types from intact mouse brain and preserves the in vivo transcriptome better than other sequencing strategies that require cell purification. Tuberous sclerosis (TSC) is characterized by activation of the mTORC1 signaling in the brain with severe neurological symptoms. Interestingly, mTORC1 activation was recently shown to cause global proximal APA shift through regulating an APA factor in non-brain cells, which suggests that aberrant APA is a previously unknown cellular phenotype of TSC. In this application, we propose to conduct a proof-of- principle study in which we apply cTag-PAPERCLIP to investigate APA in TSC by incorporating new tools that we recently developed. We will identify changes in APA isoform expression in response to acute loss of Tsc1 and activation of mTORC1 signaling by in vivo cTag-PAPERCLIP profiling in 3 brain cell types: excitatory neurons, inhibitory neurons and astrocytes. We will use our established bioinformatics pipeline to identify robust and biologically important APA changes in each cell type. We will perform RNA-seq to provide independent validations for the observed APA changes. APA can alter the cellular signaling network through production of truncated proteins or loss of 3? UTR regulation from RNA-binding proteins and microRNAs. For APA changes predicted to carry such effects, we will experimentally validate the predicted quantitative or qualitative protein changes in common cell lines and primary neuron cultures. Lastly, we will also investigate the molecular mechanisms responsible for APA changes from mTORC1 activation in primary neuron cultures. Upon completion of the proposed project, we expect to reveal for the first time how mTORC1 activation in TSC alters the cellular APA landscapes in excitatory neurons, inhibitory neurons and astrocytes in addition to addressing a key question?how APA dysregulation contributes to TSC pathogenesis. Our results will also provide new insights into the knowledge and clinical management of TSC and other neurological disorders with mTOR pathway dysregulation (?mTORopathies?)